Summary Forkhead box protein O (FoxO) family transcription factors are evolutionarily conserved proteins that play essential roles during development and in postnatal physiology in the heart. FoxOs play a dichotomous role in the regulation of cell survival/death in a context-dependent manner: they mediate cardiomyocyte (CM) cell death under some conditions but promote cell survival under other conditions. FoxOs can promote cell death by stimulating transcription of Bim, TRAIL, FasL, Bcl-6 and NOXA or lead to insulin resistance by downregulating IRS. On the other hand, endogenous FoxOs promote CM survival in response to oxidative stress and mediate the pro-survival effects of Sirt1 in CMs during ischemia/reperfusion (I/R). Moreover, FoxO1- dependent transcription of autophagy genes is required for survival of CMs during starvation. At present, however, how FoxOs mediate their dichotomous cellular functions is not well understood. Better understanding of the underlying mechanisms may make it possible to promote only the salutary function of FoxO1 while inhibiting the pro-death function of FoxO1 in the heart in the clinical setting. Our preliminary results suggest that Mst1, a pro-apoptotic kinase, can convert FoxO1 from pro-apoptotic to pro-survival through sequential phosphorylation of FoxO1 and C/EBP-? in CMs. Our overall hypothesis is that Mst1 converts the function of the transcription factor FoxO1 from pro-apoptotic to pro-survival through sequential phosphorylation of FoxO1 at the DNA binding domain (DBD) and C/EBP-??at Thr250 (mouse). We will: Aim 1 Show that FoxO1 promotes CM survival during myocardial ischemia and I/R through Mst1-induced phosphorylation of at the DBD; Aim 2 Demonstrate that Mst1 induces dissociation of FoxO1 from the promoter of pro- apoptotic FoxO target genes and association with C/EBP-? to promote transcription of pro-survival molecules; Aim 3 Demonstrate that Mst1-induced phosphorylation of C/EBP-? at Thr250 is FoxO1- dependent, and that Thr250 phosphorylation of C/EBP-? promotes transcription of pro-survival factors; Aim 4 Demonstrate that C/EBP-? phosphorylation at Thr250 is salutary and plays an essential role in mediating the salutary effect of the Mst1-FoxO1 pathway during ischemia and I/R. We will use cardiac- specific FoxO1 knockout (KO) (FoxO1cKO) and cardiac-specific C/EBP-? KO (C/EBP-? cKO) mice, as well as newly generated C/EBP-? (T250E) knock-in (KI) mice in conjunction with AAV-mediated gene delivery and proteomics. Our study will elucidate the molecular mechanism by which FoxO transcription factors regulate directionally opposite cellular functions, namely survival and death, in a coordinated manner and through interaction with Mst1 and C/EBP-?. Knowledge obtained from this study should be useful for the development of molecular interventions to convert the function of FoxO transcription factors from dichotomous to fully cardioprotective and facilitate survival of CMs in the setting of myocardial injury.